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Non-Classical Functions of Human Topoisomerase I: Genome-wide and Pharmacological Analyses

ABSTRACT: The biological functions of nuclear topoisomerase I (Top1) have been difficult to study because knocking out TOP1 is lethal in metazoans. To reveal the functions of human Top1, we have generated stable Top1siRNA cell lines from colon and breast carcinomas (HCT116-siTop1 and MCF-7-siTop1, respectively). In those cells, Top2 compensates for Top1 deficiency. A prominent feature of the siTop1 cells is genomic instability, with chromosomal aberrations and histone gamma-H2AX foci associated with replication. siTop1 cells also show rDNA and nucleolar alterations, and increased nuclear volume. Genome-wide transcription profiling revealed 55 genes with consistent changes in siTop1 cells. Among them, asparagine synthetase (ASNS) was reduced in siTop1 cells, as it also was in cells with transient Top1 downregulation. Conversely, Top1 complementation increased ASNS, indicating a causal link between Top1 and ASNS expression. Correspondingly, pharmacological profiling showed l-asparaginase hypersensitivity in the siTop1 cells. Resistance to camptothecin, aphidicolin, hydroxyurea and staurosporine, and hypersensitivity to etoposide and actinomycin D demonstrated that Top1, in addition to being the target of camptothecins, also regulates DNA replication, rDNA stability and apoptosis. Overall, our studies demonstrate the pleiotropic nature of human Top1 activities. In addition to its classical DNA nicking-closing functions, Top1 plays critical non-classical roles in genomic stability, gene-specific transcription, and response to various anticancer agents. Keywords: genetic modification design

ORGANISM(S): Homo sapiens

PROVIDER: GSE7161 | GEO | 2008/02/01

SECONDARY ACCESSION(S): PRJNA98165

REPOSITORIES: GEO

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Project description:Rationale: Fibrotic hypersensitivity pneumonitis is a debilitating interstitial lung disease driven by incompletely understood immune mechanisms. Objectives: To elucidate immune aberrations in fibrotic hypersensitivity pneumonitis in single-cell resolution. Methods: Single-cell 5’ RNA sequencing was conducted on peripheral blood mononuclear cells and bronchoalveolar lavage cells obtained from 45 patients with fibrotic hypersensitivity pneumonitis, 63 idiopathic pulmonary fibrosis, 4 non-fibrotic hypersensitivity pneumonitis, and 36 healthy controls in the United States and Mexico. Analyses included differential gene expression (Seurat), transcription factor activity imputation (DoRothEA-VIPER), and trajectory analyses (Monocle3/Velocyto-scVelo-CellRank). Measurements and Main Results: Overall, 501,534 peripheral blood mononuclear cells from 110 patients and controls and 88,336 bronchoalveolar lavage cells from 19 patients were profiled. Compared to controls, fibrotic hypersensitivity pneumonitis has elevated classical monocytes (adjusted-p=2.5e-3) and are enriched in CCL3hi/CCL4hi and S100Ahi classical monocytes (adjusted-p<2.2e-16). Trajectory analyses demonstrate that S100Ahi classical monocytes differentiate into SPP1hi lung macrophages associated with fibrosis. Compared to both controls and idiopathic pulmonary fibrosis, fibrotic hypersensitivity pneumonitis patient cells are significantly enriched in GZMhi cytotoxic T cells. These cells exhibit transcription factor activities indicative of TGFb and TNFa/NFkB pathways. These results are publicly available at https://ildimmunecellatlas.org. Conclusions: Single-cell transcriptomics of fibrotic hypersensitivity pneumonitis patients uncovered novel immune perturbations, including previously undescribed increases in GZMhi cytotoxic CD4+ and CD8+ T cells – reflecting this disease’s unique inflammatory T-cell driven nature – as well as increased S100Ahi and CCL3hi/CCL4hi classical monocytes also observed in idiopathic pulmonary fibrosis. Both cell populations may guide the development of new biomarkers and therapeutic interventions.

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Non-Classical Functions of Human Topoisomerase I: Genome-wide and Pharmacological Analyses

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